Normal reproduction and the maintenance of life in all mammalian species requires complex patterns of steroidogenic activities. In this renewal application it is proposed to elucidate in biochemical terms the multifactorial regulation of steroid hydroxylase gene expression in the adrenal cortex. During the past granting period we have investigated three levels of regulation of expression of these genes (maintenance of optimal steroidogenic capacity regulated in part by ACTH via cAMP, tissue-specific, and developmental) using cDNA probes specific for the various steroid hydroxylase mRNAs. In the coming granting period we propose to establish at the transcriptional level, the trans-acting factors and cis-regulatory sequences involved this complex pattern of regulation of steroid hydroxylase gene expression. Utilizing genes encoding human and bovine P-450 17 alpha human and bovine P-450scc' human P-450C21 and bovine adrenodoxin we will identify adrenal nuclear proteins which transcription of these genes in response to cAMP (so-called SHIPs) and the cis-sequences to which they bind. A combination of gel retardation, DNA footprinting, CAT activities and in vitro transcription assays will be used to identify these factors followed by their purification and characterization. In addition we will identify similar factors which are required for the fetal imprinting of steroid hydroxylase genes in the fetal bovine adrenal cortex using the same techniques. Also we will determine the molecular basis of the cell specific regulation of P-450 17 alpha in bovine fasiculata-reticularis compared to zona glomerulose as well as the reason for the absence of expression of P-450 17 alpha in rat adrenal cortex. Likewise the cell specific factors which regulate adrenodoxin gene expression in adrenal cortex, liver and kidney will be identified. Finally utilizing mutant regulatory subunits of cAMP-dependent protein kinase we will establish whether this enzyme plays a role in steroid hydroxylase gene expression. In summary, during the proposed 5 years we will identify, characterize and compare trans-acting factors which regulate adrenal steroid hydroxylase gene expression at the hormone mediated, tissue-specific and developmental levels. The results of these studies will permit assessment of both the complexity and the relatedness of these processes. Furthermore they will show the way to a new set of genes, those encoding trans-acting factors which regulate steroid hydroxylase gene expression.